Non essential amino acids (neaa)

MDCK cells were obtained from the American Type Culture Collection (ATCC). MDCK cells were maintained at 37 °C with 5% CO2 in DMEM medium (GIBCO), supplemented with 10% FCS (GIBCO), l-glutamine (GIBCO), and nonessential amino acids (GIBCO). HEK 293T cells were obtained from the ATCC and maintained in DMEM medium, supplemented with 10% FBS, 1 mM sodium pyruvate, and nonessential amino acids. Mouse fibroblast cell line L929 (ECACC; L929 (NCTC); #85103115) was purchased from Sigma-Aldrich (Taufkirchen, Germany). The L929 subclone 15.9, which is highly susceptible to the mouse-adapted scrapie strain 22L, was used for all experiments^{29 (link),30 (link)}. CAD5 cells^{31 (link)} were a kind gift of Corinne Lasmezas (The Scripps Research Institute, FL).

HeLa (ATCC), T-REx HeLa (Invitrogen) and HCT116 cells (ATCC) were cultured in Minimum Essential Medium (Sigma) supplemented with 10% FBS, 1% penicillin-streptomycin (Invitrogen), and non-essential amino acids (Invitrogen). A549 (ATCC) cells were cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin, and non-essential amino acids. For S phase synchronization, HeLa cells were treated twice with 2 mM thymidine (Sigma) for 16 h.

Mouse neuroblastoma 2a (Neuro 2a) cells (ATCC^®, CCL-131, Manassas, VA, USA) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, and 1% non-essential amino acids (all Thermo Fisher Scientific, Rockford, IL, USA) at 37 °C in a humidified atmosphere with 5% CO2. For coimmunoprecipitation assay, ~3,000,000 cells were seeded in one 100 mM plate. For the cell surface biotinylation assay, ~800,000 cells were seeded in 60 mM plates. For the live and fixed cell imaging, including FRET, ~25,000 cells were seeded in 35 mM glass-bottom dishes (MatTek Corporation, Ashland, MA, USA) or 24-well plates. Half an hour prior to the live microscopy assays, cells were transferred into DMEM lacking phenyl red.
Neuro 2a cells were transiently transfected with Effectene™ Transfection Reagent Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s guidelines. Cells were double transfected with a total of 4000 ng or 1200 ng of DNA for each 100 mM or 60 mM plate, respectively. For 35 mM glass-bottom dishes and 24-well plates, cells were transfected with 200 ng for single transfections and 400 ng for double transfections. All of the experiments were performed 48 h post-transfection.

GBM cell lines were purchased from American Type Culture Collection (ATCC, USA) and grown in a humidified, 5 % CO2 incubator at 37 °C in the complete media as described by the provider. DBTRG-05MG (ATCC, USA, #CRL-2020) cells were grown in ATCC-formulated RMPI-1640 medium (Gibco, Life Technologies, USA, #A10491-01) supplemented with 10 % foetal bovine calf serum (Gibco, Life Technologies, USA, #16000044), non-essential amino acids (Gibco, Life Technologies, USA, #11140050) and 100 U/mL penicillin, 0.1 mg/mL streptomycin (Gibco, Life Technologies, USA, #15140122) to generate complete media. M059J (ATCC, USA, #CRL-2366) cells were grown in media containing a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Medium (DMEM-F12, ATCC, USA, #30-2006) supplemented with 10 % foetal bovine calf serum, non-essential amino acids and 100 U/mL penicillin, 0.1 mg/mL streptomycin. LN18 (ATCC, USA, #CRL-2610) and LN229 (ATCC, USA, #CRL-2611) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC, USA, #30-2002) supplemented with 5 % foetal bovine calf serum and 100 U/mL penicillin, 0.1 mg/mL streptomycin. HeLa cells were grown in a humidified, 5 % CO2 incubator at 37 °C in the complete media, Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC, USA, #30-2002) supplemented with 10 % foetal bovine calf serum and 100 U/mL penicillin, 0.1 mg/mL streptomycin.

In this study, we used MDA-MB-231 (hormonal-independent triple-negative breast cancer). Cells were cultured in DMEM-F12 (Biowest), consisting of 10% fetal bovine serum (Biowest), 1% antibiotic-antimycotic (ATCC), and 0.5% non-essential amino acids (GIBCO) in a humidified incubator at 37°C, in the presence of 5% CO₂. When the mixture had 80% confluency, the cells were separated with Versene (KCl, EDTA, NaCl, and Tris-HCl), and cell viability was determined through a Trypan blue assay (4%) with a Neubauer camera (Strober, 2001 (link)).